ISOLATION AND IDENTIFICATION OF METALLOTHIONEIN ISOFORMS (MT-1 AND MT-2) IN THE RAT TESTIS

It has been a long-lasting controversial issue as to whether or not th e male genital organs, such as the testis and prostate, contain metall othioneins (MTs), a group of cysteine-rich heavy-metal-binding protein s that play a role in detoxifying heavy metals such as cadmium (Cd). E arlier studies reported that the rodent testis lacks MTs and concluded that this is why the testis is very susceptible to Cd, although other indirect experimental evidence suggests that MTs are present in this organ. A deficiency of MTs in the testis was originally suspected on t he basis of amino acid composition analysis, since MT-like proteins is olated as Cd-binding proteins did not have a characteristic MT structu re. In the present study, we demonstrate that the fat testis indeed ex presses Cd-binding proteins with sequences identical to those previous ly described for MT-I and MT-2, the major isoforms. To confirm that MT -I and MT-2 are present in the rat testis, we purified and isolated Cd -binding proteins by homogenization using Cd-containing buffer. follow ed by sequential purification using Sephadex G-75 gel filtration chrom atography and anion HPLC column chromatography, which yielded Cd-bindi ng protein-1 (Cd-BP-1) and -2 (Cd-BP-2). After pyridylethylation, Cd-B P-1 and Cd-BP-2 were subjected to specific protein fragmentation by ac ids and endopeptidases, which revealed that these Cd-binding proteins have the same primary structures as MT-1 and MT-2 respectively. Thus w e believe that the present results clearly resolve the long-standing d ebate about the presence of MTs in the testis, at least in the rodent.